These studies were aimed at characterization of the murine immune response to the mucosal pathogen, Chlamydia trachomatis. Knowledge of the cellular and molecular mechanism(s) utilized by the murine host to clear chlamydial infections is required for design and development of a successful chlamydial vaccine. Using a panel of inbred mouse strains bearing targeted mutations in a variety of immunologically relevant genes (gene knockout mice), we found that immunity to chlamydial infection of the genital mucosa required the presence of functional genes encoding the ab T cell receptor molecule and the cytokines IL-12, IFN-g and TNF- a. Genes encoding the gd T cell receptor, IL-6, and IL-10 were not required. These data indicated that immunity to Chlamydia is mediated by traditional IL-12-driven type 1 CD4+ T cells secreting IFN-g and TNF-a. The molecular mechanism by which these cells eliminate Chlamydia from infected epithelial cells did not appear to involve Fas-mediated apoptosis or the pore forming protein, perforin, since mice lacking the Fas or perforin genes cleared infections as efficiently as controls. Comparison of distinct C. trachomatis isolates revealed variation in their sensitivity to the type I cytokine, IFN-g. Both in vivo and in vitro, IFN-g inhibited the growth of human C. trachomatis serovars A through K but not the murine strain, mouse pneumonitis (MoPn). IFN-g-mediated inhibition of human chlamydial growth in vitro occurred by chlamydiacidal rather than chlamydiastatic mechanisms since chlamydial growth did not resume following removal of the inhibiting cytokine. In murine cells, stimulation of inducible nitric oxide synthase (iNOS) provides one potential mechanism of IFN-g action. However, the finding of normal chlamydial clearance in iNOS deficient mice and the inability of an iNOS inhibitor to reverse IFN-g-mediated inhibition in vitro argued against a significant role for this pathway. Thus, the molecular mechanism whereby IFN-g irreversibly limits chlamydial growth remains to be determined. Infections with human or murine C. trachomatis strains were marginally inhibited in the absence of TNF-a, another type 1 cytokine produced during infection. TNF- a-mediated inhibition could not be reproduced in vitro, however, suggesting that the action of this cytokine is indirect involving cells and/or mediators not present in the in vitro culture system. These findings indicate that the successful Chlamydia vaccine will recruit IFN-g-secreting T cells to the site of mucosal infection. They also indicate that use of MoPn as a model system for vaccine development and testing may be inappropriate since MoPn is relatively IFN-g-insensitive, although other aspects of type 1 CD4+ T cell-mediated immunity are probably important in MoPn resistance. Combined with data defining the trafficking of lymphocytes to Chlamydia-infected mucosae (Z01-AI-00771-02-LICP), these studies provide a logical theoretical basis for the development and delivery of an efficacious C. trachomatis vaccine.